Heme oxygenase-1 fused to a TAT peptide transduces and protects pancreatic β-cells

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Abstract

Transplantation of islets is becoming an established method for treating type 1 diabetes. However, viability of islets is greatly affected by necrosis/apoptosis induced by oxidative stress and other insults during isolation and subsequent in vitro culture. Expression of cytoprotective proteins, such as heme oxygenase-1 (HO-1), reduces the deleterious effects of oxidative stress in transplantable islets. We have generated a fusion protein composed of HO-1 and TAT protein transduction domain (TAT/PTD), an 11-aa cell penetrating peptide from the human immunodeficiency virus TAT protein. Transduction of TAT/PTD–HO-1 to insulin-producing cells protects against TNF-α-mediated cytotoxicity. TAT/PTD–HO-1 transduction to islets does not impair islet physiology, as assessed by reversion of chemically induced diabetes in immunodeficient mice. Finally, we report that transduction of HO-1 fusion protein into islets improves islet viability in culture. This approach might have a positive impact on the availability of islets for transplantation.

Section snippets

Cloning and related techniques

The recombinant TAT/PTD anti-apoptotic protein construct was generated by inserting the DNA coding region of murine heme oxygenase-1 (HO-1) (produced by Robert Oliver University of Miami) in the EcoRI site of the pTAT expression vector (kind gift from Dr. Steven Dowdy from USDC).

Protein generation and purification

The isolation and purification of TAT/PTD–HO-1 and HO-1 (no TAT) were done as previously described [11] with some modifications in the washing steps: briefly, the Ni–NTA column, with bacterial extract, was sequentially

Generation and purification of recombinant TAT/PTD–HO-1 fusion protein

To generate a TAT/PTD–HO-1 recombinant protein we inserted the coding region of the murine heme oxygenase gene in-frame with the 11-amino-acid PTD of the HIV/TAT protein in the pTAT-HA expression vector. Bl 21 Escherichia coli was used to produce the recombinant TAT/PTD–HO-1 protein. A green coloration of the bacterial culture was consistently observed, suggesting that the recombinant TAT/PTD–HO-1 metabolized the heme from E. coli into biliverdin, that accumulated due to the lack of biliverdin

Conclusions

Our data show that transduction of insulin producing cells with a recombinant HO-1 protein fused to the TAT/PTD confers cytoprotection against TNF-α-cytotoxicity and cell death during culture. Transduction with TAT/PTD–HO-1 protein might be useful to confer transient cytoprotection and therefore enhance the viability of transplantable islets. Prolonged culture of viable islets could help develop immunosuppressive regimes for recipients prior to islet transplantation.

Acknowledgments

This work was supported by a grant from the National Institutes of Health (DK-59993) awarded to R.L.P., the Diabetes Research Institute Foundation (Hollywood, FL), and the Foundation for Diabetes Research.

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